A major component of barrier function in stratified squamous epithelia is the cornified cell envelope (CE). This is a multi-component 10 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid envelope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the cross-linking of several structural by transglutaminases. Studies on the biology and assembly of the protein and lipid components are a major effort of this laboratory. Specifically, we are studying: (i) the cross-linking of proteins in CEs isolated from a variety of sources to explore which proteins are cross-linked together through which glutamines and lysines, and to provide information on structure and function; (ii) several key structural proteins such as loricrin, the small proline rich protein (SPR) families, involucrin, envoplakin and periplakin; (iii) the ceramide lipids which become covalently attached to the CE; (iv) the earliest stages of CE assembly produced in cultured keratinocytes; and (v) an attempt to recreate a CE-like structure using an in vitro synthetic lipid vesicle (slv) model system. CE protein envelope structure and assembly We are studying the features of CEs isolated from several sources, including human epidermis, cultured human epidermal keratinocytes induced to terminally differentiate, human oral epithelia, human uterine fluid material, and mouse inner root sheaths. We have used controlled proteolysis to dissect apart the CEs, separate cross-linked peptides, and perform protein sequencing. Together, our data are consistent with the possibility that CE assembly is initiated along the plasma membrane at interdesmosomal sites by head-to-head and head-to-tail cross-linking of involucrin to itself, and perhaps to envoplakin and periplakin. Shortly later, involucrin deposition spreads to desmosomal sites so that a continuous layer of involucrin, envoplakin and perhaps periplakin is formed along the cell periphery: this layer perhaps forms a scaffold for the later stages of CE assembly involving substantial deposition of other proteins such as loricrin and SPRs. Loricrin We have expressed human loricrin in bacteria and used it to characterize its structure, biochemical properties, and cross-linking by epidermal transglutaminases (TGase) in vitro. By biophysical measurements it has some structure in solution associated with its multiple tyrosines. It is a complete TGase substrate because it is oligomerized by all three epidermal TGases in in vitro reactions, although with different kinetic efficiencies, and utilization of different glutamines and lysines. From comparisons of the residues used in vitro with those used in vivo from sequencing of CEs, we can conclude that both TGase 1 and TGase 3 are required for the correct cross-linking of loricrin in vivo. We have found that following cross-linking in vitro, loricrin becomes a compact near-spherical in shape and far more soluble. From this we can speculate that epidermal CEs contain about two layers of loricrin molecules. Further, the data suggest that preliminary cross-linking by the transglutaminase 3 enzyme may render the protein more soluble to allow transloaction to the cell periphery where it is eventually attached to the growing CE structure. The proximal promoter of the human loricrin gene resides within the first 160 bp above the transcription initiation start site. Our data indicate that there are multiple positively- and negatively-interacting elements which confer tight epidermal specific expression on the loricrin gene during various stages of epidermal differentiation. Small proline rich proteins SPRs consist of four distinct families consisting of from one to 11 members. We have expressed one member of each of the human SPR1, SPR2 and SPR3 proteins for in vitro studies. By circular dichroism, they have little organized structure in solution. What structure is present can be attributed to the central proline-rich peptide repeats, and the signal strength is proportional to the number of repeats. The SPR proteins are also complete substrates in in vitro cross-linking reactions for the three TGases commonly expressed in the epidermis. In all cases of SPR proteins studied, the glutamines and lysines used for cross-linking are located only on the end domains, suggesting they may function as cross-bridging proteins. However, the details are different, which have provided a wealth of information on how the proteins may be used in tissues in vivo. Solution nmr structural studies on the SPR2 and 3 proteins have been performed. Unfortunately, these proteins have little organized structure in solution and only short range interactions were obtained. Nevertheless, the data suggest the central peptide repeat domains adopt novel omega-loop-like protein folds. We have explored the expression of the SPR1 and SPR2 families in mouse epithelia by use of immunocytochemistry, in situ hybridization and RT-PCR. Both families are differentially expressed in different epithelia. In the case of SPR1, the amount expressed in the epidermis varies widely with the site, from which we can conclude that the amounts of SPRs may influence the biomechanical properties of the epidermal body sites. Involucrin We have expressed and purified full-length human involucrin in bacteria and baculovirus systems and showed that it retains some but not all of its expected a-helical content. We have been successful in forming small crystals, minimally useful for Xray diffraction analyses, but improved conditions and larger crystals will be needed now to solve its three-dimensional structure. In addition, various modeling analyses have predicted that the central repeat motifs of members of the small proline rich family might associate with the repeat domain of involucrin. This model has several attractive features, in that it can explain the known in vivo and in vitro biochemical and cross-linking properties of both proteins. Experiments are in progress to test this possibility. Periplakin and Envoplakin These members of the plakin family are intimately involved in the earliest stages of CE assembly in epithelia. We have direct evidence that sequences on their tails are involved in cross-linking to keratin filaments, and other CE proteins, as well as attachment of ceramide lipids. In order to explore this further, we have expressed portions of the rods, tails and rods + tails of each protein. Crystallization trials have been set up for periplakin in an effort to obtain three-dimensional structural information. Whereas periplakin alone is very soluble in physiological buffers, envoplakin is highly insoluble, but when mixed together, periplakin stabilizes envoplakin into a soluble oligomer (probably heterotetramer). Further, we have found that only intact forms of these two proteins bind to synthetic lipid vesicles and in a calcium dependent manner. Our data suggest that as soon as these proteins are expressed in epithelial cells, they will preferentially oligomerize and associate with plasma membranes. Further studies with transglutaminase cross-linking with synthetic lipid vesicles to investigate their possible roles in the earliest stages of CE assembly.